A formidable toolkit exists for manipulating protein expression at the transcriptional level, but the methods for post-translational modulation are few and largely rely on the expression of fusion proteins. While such systems can provide important insights into cellular processes, the necessity of fusion protein expression can be constraining and the potential for therapeutic use is nil. A small molecule strategy to induce the degradation of endogenous proteins would clearly be a tremendously useful tool for probing protein function and an exciting new approach for chemotherapy. The challenge is to develop technology that is widely applicable.
Targeted gene knockout and RNAi are currently the methods choice for removing endogenous proteins. Both methods have been enormously useful, but both have serious limitations. The proteins of greatest interest are often essential and therefore not amenable to gene knockout. While this problem can often be circumvented with regulated gene expression or conditional gene targeting (e.g., the tetracycline and Cre-loxP systems), such experiments can be difficult to interpret because of the long time between loss of gene expression and protein depletion. RNAi is also limited by the time of induction, which can be many days. RNAi knockdown can often be incomplete or beset with off-target effects. Importantly, not all organisms have RNAi pathways: it is notably absent in the malaria parasite. More seriously, major hurdles in permeability and delivery will need to be surmounted before this method can have widespread use in mammals and in the clinic. These issues might be avoided if the intracellular protein degradation machinery could be exploited via a small molecule.
Several laboratories have developed systems where small molecules regulate protein degradation. The most common strategy involves expression of fusion proteins that couple the target to an unstable “degron” domain. The degron domain is stabilized by the presence of a ligand; removal of the ligand induces degradation. For example, unstable DHFR mutants have been used to create a temperature sensitive degron that is stabilized by methotrexate or trimethoprim (TMP). Other systems use degrons based on the rapamycin interacting proteins FKBP12 and FKBP-rapamycin binding protein (FRB). These domains are unstable in the absence of rapamycin-based ligands; again degradation is induced when the ligand is removed. A particularly clever use of the rapamycin-based dimerization is found in the SURF system. In this case, rapamycin stops degradation by causing the degron to be removed from the target protein. These systems have proven very useful, but are limited by the requirement for the constant presence of ligand to maintain protein levels—a system that induced degradation by addition of a small molecule would be easier to maintain. Another approach uses a pair of fusion proteins to localize a target protein directly to the proteasome, with one partner on the target protein and a second on a proteasome subunit. Importantly, the success of this strategy demonstrates that proteasome localization is sufficient to induce degradation. A similar strategy has been used to target protein degradation in bacteria. The systems described above modulate levels of transgenic fusion proteins with varying degrees of success, but cannot be used to reduce the levels of endogenous proteins.
Proteolysis targeting chimeric molecules (PROTACs) have been described. PROTACs contain a ligand that recognizes the target protein linked to a ligand that binds to a specific E3 ubiquitin ligase. Degradation of methionine aminopeptidase, androgen receptor, estrogen receptor and the aryl hydrocarbon receptor have been reported. In most cases, the E3 ligase-targeting ligand is a peptide, which limits therapeutic use. In addition, this method will only work in cells and organisms that express the targeted E3 ligase. More generally, the ubiquitin pathways are extremely complicated and poorly understood, which suggests that controlled manipulation of these pathways difficult. A small molecule strategy that targets proteins directly to the proteasome would have many advantages over PROTACs.
Additionally, proteins must fold into their correct three-dimensional conformation to achieve their biological function. The native conformation of a polypeptide is encoded within its primary amino acid sequence, and even a single mutation in an amino acid sequence can impair the ability of a protein to achieve its proper conformation and/or function. When proteins fail to fold correctly, or are not active, the biological and clinical effects can be devastating. For example, protein aggregation and misfolding are primary contributors to many human diseases, such as autosomal dominant retinitis pigmentosa, Alzheimer's disease, α1-antitrypsin deficiency, cystic fibrosis, nephrogenic diabetes insipidus, and prion-mediated infections. In other protein-folding disorders, such as age-related macular degeneration, Parkinson's disease, and Huntington's disease, pathology results because of the cytotoxic effects of the misfolded protein.
Mutant (e.g., misfolded) proteins are often recognized by the endoplasmic reticulum (ER) quality control system and targeted for degradation by the proteasome. Besides the proteasomal pathway, autophagy is another major cellular mechanism for protein degradation. While autophagy can be stimulated by a variety of intracellular and extracellular stresses, including amino-acid starvation, aggregation of protein, and accumulation of damaged organelles, autophagy appears to be a largely non-selective process. Aggregate-prone polyglutamine and polyalanine expanded proteins associated with Huntington's disease are degraded by autophagy, and inhibition of autophagy reduces the toxicity of mutant Huntington proteins in fly and mouse models of Huntington disease. Autophagy has also been shown to contribute to the elimination of proteins accumulated in the ER. Methods for increasing the degradation of mutated proteins might enhance the elimination of such proteins, thereby decreasing or eliminating their cytotoxic effects. See, for example, United States Patent Application No. 2010/0087474 A1 entitled “Materials and Methods for Enhanced Degradation of Mutant Proteins Associate with Human Disease,” which is hereby incorporated by reference in its entirety. A small molecule strategy to induce the degradation of mutant proteins would also be a useful tool.